Particulate coated hydrogel microparticles

ABSTRACT

A composition includes a plurality of particulate coated hydrogel microparticles, each of the microparticles including a hydrogel inner core and a particulate shell defined by a plurality of solid nanoparticles, the particulate shell inhibiting aggregation of the microparticles in an aqueous medium and being permeable to allow release of agents from the hydrogel inner core.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.62/650,661, filed Mar. 30, 2018, the subject matter of which isincorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No.R01AR063194 and T32HL134622, awarded by The National Institutes ofHealth. The United States government has certain rights to theinvention.

BACKGROUND

Gelatin hydrogel spheres at nano or micron sizes have been widely usedas carriers for tissue engineering and drug delivery purposes. As aderivative from collagen, gelatin molecule contains abundant integrinbinding sites that promote cell growth and functioning. Gelatin carriersalso bear strong capability of adsorbing and releasing a variety of drugmolecules with different sizes, hydrophobicities, electrical propertiesor affinities. For most of the applications, gelatin microspheres needto be homogeneously suspended prior to use. For example, whenintegrating gelatin microspheres to densely packed cells for tissueconstruct generation, gelatin microspheres are required to be mixed withcells for 3 dimensional (3D) constructions. However, due to strongintermolecular interactions, such as electrostatic interaction, afterbeing placed into aqueous solutions, gelatin microspheres tend toaggregate. It is quite challenging to homogenously suspend gelatinmicrospheres in aqueous solution or mix them with cells. Tediouspipetting is usually performed to reduce the aggregation effect. Suchproblem greatly hampers the advantages of gelatin carrier in large scaleuse.

Traditional methods to overcome the problem of gelatin microspheresaggregation include crosslinking and coating with shells. In theory,crosslinking reduces intermolecular interactions of gelatin moleculesthrough networking nearby molecules. In practice, crosslinking does notwell prevent gelatin microspheres from aggregation. Alternatively,coating gelatin microspheres with organic and inorganic materialsprevent interactions among gelatin molecules. However, for the deliveryof drugs including small molecules and macromolecules, shells inhibitthe penetration of payloads, resulting in insufficient loading and/orreleasing.

SUMMARY

Embodiments described herein relate to compositions that can be used forbioactive agent delivery and tissue engineering, and more particularlyto compositions that can provide delivery of bioactive agents in atemporally controlled or predetermined manner to cells or tissue in avariety of biomedical applications, including tissue engineering, drugdiscovery applications, and regenerative medicine.

In some embodiments, the composition can include a plurality ofparticulate coated hydrogel microparticles or microspheres. Each of themicroparticles can include a hydrogel inner core and a porousparticulate shell defined by a plurality of solid nanoparticles. Theparticulate shell can inhibit aggregation of the microparticles in anaqueous medium. Optionally, the hydrogel inner core can include one ormore bioactive agents, and the particulate shell can allow release ofthe bioactive agents from the hydrogel inner core in a sustained,controlled, and/or predetermined manner.

The particulate coated hydrogel microparticles can be formed using areverse Pickering emulsion process that assembles the solid particlesonto outer surfaces of hydrogel microparticles of an emulsion.Advantageously, it was found that particulate coated hydrogelmicroparticles, which include a gelatin core and nanoparticle shell canoutperform traditional gelatin microparticles both in supporting cellfunctions and releasing drugs. In addition, due to its ability of easilysuspending in aqueous solution, the particulate coated hydrogelmicroparticles can be readily adopted for large scale production oftissue constructs for tissue engineering applications and for generatingcell aggregates for in vitro testing of pharmaceutical agents.

In some embodiments, the microparticles can have an average diameter ofabout 1 μm to about 500 μm, and the nanoparticles can have an averagediameter of about 50 nm to about 900 nm.

In other embodiments, the hydrogel inner core can include gelatin. Thegelatin can be optionally cross-linked to control degradation of theinner core and/or diffusion or release of the bioactive agent from theinner core.

In some embodiments, the plurality of solid nanoparticles can includesolid inorganic nanoparticles, such as silica nanoparticles.

The composition can also include a plurality of cells. The cells can beuniformly dispersed with the microparticles in the composition. Thecells can include progenitor cells and stem cells, such as mesenchymalstem cells and cancer cells, and the composition can be used in tissueengineering application and drug discovery applications.

Other embodiments described herein relate to a method of a forming aplurality of particulate coated hydrogel microparticles. The methodincludes suspending a plurality of solid nanoparticles in an organicsolvent. Hydrogel forming natural polymer macromers are dissolved in anaqueous solution. The aqueous solution of the hydrogel forming naturalpolymer macromers is added to the organic solvent containing the solidnanoparticles to form a Pickering emulsion. The Pickering emulsionincludes a plurality of uniformly dispersed microparticles of thehydrogel forming polymer macromers and the solid nanoparticles coatingouter surfaces of the hydrogel forming polymer macromers. The hydrogelforming polymer macromers are then solidified to form particulate coatedhydrogel microsphere. The particulate coated hydrogel microparticles arethen isolated from the organic solvent.

In some embodiments, particulate coated hydrogel microparticles formedby the method include a hydrogel inner core and a porous particulateshell defined by a plurality of solid nanoparticles. The particulateshell can inhibit aggregation of the microparticles in an aqueous mediumand be permeable to allow release or diffusion of bioactive agentscontained in the hydrogel inner core from the hydrogel inner core.

In other embodiments, the hydrogel inner core includes at least onebioactive agent. The at least one bioactive agent can be added to thehydrogel inner core during or after formation of the particulate coatedhydrogel microparticles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic showing the preparation of gelatincore/particle shell microspheres.

FIGS. 2(A-D) illustrate images showing core/shell microspheres havebetter water suspension ability. (A) Silica nanoparticles used for theassembly. Bar: 500 nm (B) 2 mg Microspheres in 1 mL water after 5 svortexing. Left: Core/shell microspheres; Right: Gelatin microsphereswithout silica nanoparticles. Arrows indicate visibly aggregatedmicrospheres. (C) Microscopic image shows 2 mg Core/shell microspheresin 1 mL water after 5 s vortexing. (D) Microscopic image shows 2 mggelatin microspheres without silica shell in 1 mL water after 5 svortexing. Bars: 200 μm.

FIGS. 3(A-C) illustrate LSCM images of silica shell/gelatin coremicrospheres. Microspheres are squeezed by cover slips for imaging ofsurface coats. (A) Large scale image shows core/shell structure. Bar:100 μm. (B) Z-stack LSCM image shows surface of microspheres. (C) Samez-stack LSCM image shows center of microspheres. Bars: 50 μm. Green:FITC labeled silica; Red: TRITC labeled gelatin.

FIG. 4 illustrates graphs showing GAG and DNA content analysis of cellaggregates with exogenous TGF-β1 supply after 1 week or 2 weeks.

FIG. 5 illustrates graphs showing GAG and DNA content analysis of cellaggregates with endogenous TGF-β1 supply after 1 week or 2 weeks.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thepresent invention pertains. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Edition, Springer-Verlag: NewYork, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994.The definitions provided herein are to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present invention.

The term “antisense” nucleic acid refers to oligonucleotides whichspecifically hybridize (e.g., bind) under cellular conditions with agene sequence, such as at the cellular mRNA and/or genomic DNA level, soas to inhibit expression of that gene, e.g., by inhibiting transcriptionand/or translation. The binding may be by conventional base paircomplementarily, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix.

The term “bioactive agent” refers to any agent capable of promotingtissue formation, destruction, and/or targeting a specific disease state(e.g., cancer). When administered to a host, both human and animal,e.g., the bioactive agent may be used as part of a prophylatic ortherapeutic treatment. Examples of bioactive agents can include, but arenot limited to, chemotactic agents, various proteins (e.g., short termpeptides, bone morphogenic proteins, collagen, glycoproteins, andlipoprotein), cell attachment mediators, biologically active ligands,integrin binding sequence, various growth and/or differentiation agentsand fragments thereof (e.g., epidermal growth factor (EGF), hepatocytegrowth factor (HGF), vascular endothelial growth factors (VEGF),fibroblast growth factors (e.g., bFGF), platelet derived growth factors(PDGF), insulin-like growth factor (e.g., IGF-I, IGF-II) andtransforming growth factors (e.g., TGF-β I-III)), parathyroid hormone,parathyroid hormone related peptide, bone morphogenic proteins (e.g.,BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13, BMP-14), transcriptionfactors, such as sonic hedgehog, growth differentiation factors (e.g.,GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP52 and theMP-52 variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-1,CDMP-2, CDMP-3), small molecules that affect the upregulation ofspecific growth factors, tenascin-C, hyaluronic acid, chondroitinsulfate, fibronectin, decorin, thromboelastin, thrombin-derivedpeptides, heparin-binding domains, heparin, heparan sulfate,polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interferingRNA molecules, such as siRNAs, oligonucleotides, proteoglycans,glycoproteins, glycosaminoglycans, and DNA encoding for shRNA. Inaddition, biological entities, such as viruses, virenos, and prions areconsidered bioactive agents. The bioactive agents may be water-solubleor water-insoluble and may include those having a high molecular weight,such as proteins, peptides, carbohydrates and glycoproteins.

The term “biocompatibility” or “biocompatible” when used in relation tomicroparticles or microspheres described herein refers to microparticlesor microspheres that are neither themselves toxic to the host (e.g., ananimal or human), nor degrade (if at all) at a rate that producesbyproducts at toxic concentrations in the host. To determine whether anysubject microparticles are biocompatible, it may be necessary to conducta toxicity analysis. Such assays are well known in the art.

The term “biodegradable” refers to those embodiments in whichmicroparticles or hydrogels described herein are intended to degradeduring use. In general, degradation attributable to biodegradabilityinvolves the degradation of a microsphere or hydrogel into itsconstituents and encapsulated materials. The degradation rate of abiodegradable microsphere or hydrogel often depends in part on a varietyof factors, including the identity of any constituents that form themicrosphere and hydrogel and their ratio, the identity and loading ofany material (including bioactive agent encapsulated in a microsphere),how any microsphere may be crosslinked and to what extent. For example,a microsphere that is crosslinked will, in all likelihood, degrade moreslowly than one that is not crosslinked.

The term “cell” can refer to any progenitor cell, such as totipotentstem cells, pluripotent stem cells, and multipotent stem cells, as wellas any of their lineage descendant cells, including more differentiatedcells. The terms “stem cell” and “progenitor cell” are usedinterchangeably herein. The cells can derive from embryonic, fetal, oradult tissues. Exemplary progenitor cells can be selected from, but notrestricted to, totipotent stem cells, pluripotent stem cells,multipotent stem cells, mesenchymal stem cells (MSCs), hematopoieticstem cells, neuronal stem cells, hematopoietic stem cells, pancreaticstem cells, cardiac stem cells, embryonic stem cells, embryonic germcells, neural crest stem cells, kidney stem cells, hepatic stem cells,lung stem cells, hemangioblast cells, and endothelial progenitor cells,cancer stem cells. Additional exemplary progenitor cells are selectedfrom, but not restricted to, de-differentiated chondrogenic cells,chondrogenic cells, cord blood stem cells, multi-potent adult progenitorcells, myogenic cells, osteogenic cells, tendogenic cells,ligamentogenic cells, adipogenic cells, and dermatogenic cells.

When used with respect to the bioactive agent, the term “controlledrelease” is intended to mean that the bioactive agent is released overtime in contrast to a bolus type administration in which the entireamount of the bioactive agent is presented to the target at one time.The release will vary as explained below.

The term “gene” or “recombinant gene” refers to a nucleic acidcomprising an open reading frame encoding a polypeptide, including bothexonic and (optionally) intronic sequences.

The term “gene construct” refers to a vector, plasmid, viral genome orthe like which includes an “coding sequence” for a polypeptide or whichis otherwise transcribable to a biologically active RNA (e.g.,antisense, decoy, ribozyme, etc.), can transfect cells, preferablymammalian cells, and can cause expression of the coding sequence incells transfected with the construct. The gene construct may include oneor more regulatory elements operably linked to the coding sequence, aswell as intronic sequences, poly adenylation sites, origins ofreplication, marker genes, etc.

The term “host cell” or “target cell” refers to a cell transduced with aspecified transfer vector. The cell is optionally selected from in vitrocells such as those derived from cell culture, ex vivo cells, such asthose derived from an organism, and in vivo cells, such as those in anorganism.

The term “incorporated” or “encapsulation,” when used in reference to abioactive agent or other material and a microsphere, denotes formulatinga bioactive agent or other material into a microsphere useful forcontrolled release of such agent or material. As used herein, thoseterms contemplate any manner by which a bioactive agent is incorporatedinto a microsphere, including for example: distributed throughout thematrix, appended to the surface of microparticles, and encapsulatedinside the matrix or microparticles. The term “coincorporation” or“coencapsulation” as used herein refers to the incorporation of abioactive agent in a microsphere and at least another bioactive agent orother material.

The term “microspheres”, “microdroplets”, or “microparticles” are usedinterchangeably and refer to substantially spherical structures formedby a coacervation process. The microdroplets generally have amatrix-type structure, and can incorporate and/or encapsulate abioactive agent within the matrix. The microparticles generally have asize distribution within the range of from about 1 μm to about 500 μm.In certain embodiments, over 90% of the microparticles formed in asingle preparation of microparticles have a diameter in excess of about5 μm. Other sizes are also contemplated herein

When a large number of microparticles are formed, they may have avariable size. In certain embodiments, the size distribution may beuniform, e.g., within less than about a 20% standard deviation of themedian volume diameter, and in other embodiments, still more uniform orwithin about 10% of the median volume diameter.

The term “modulation” refers to both up regulation (i.e., activation orstimulation) and down regulation (i.e., inhibition or suppression) of aresponse.

The term “nucleic acid” refers to polynucleotides, such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single-stranded (such assense or antisense) and double-stranded polynucleotides. Exemplarynucleic acids for use in the subject invention include antisense, decoymolecules, recombinant genes (including transgenes) and the like.

The phrases “parenteral administration” and “administered parenterally”means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intra-articulare, subcapsular, subarachnoid,intraspinal and intrasternal injection and infusion.

The phrase “pharmaceutically acceptable” refers to those microparticlesand dosages thereof within the scope of sound medical judgment, suitablefor use in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The term “prophylactic or therapeutic” treatment refers toadministration to the host of the subject micro and/or nanodroplets. Ifit is administered prior to clinical manifestation of the unwantedcondition (e.g., disease or other unwanted state of the host animal)then the treatment is prophylactic, i.e., it protects the host againstdeveloping the unwanted condition, whereas if administered aftermanifestation of the unwanted condition, the treatment is therapeutic(i.e., it is intended to diminish or ameliorate the existing unwantedcondition or side effects therefrom).

The terms “protein,” “polypeptide” and “peptide” are usedinterchangeably when referring to a gene product.

“Recombinant host cells” refers to cells which have been transformed ortransfected with vectors constructed using recombinant DNA techniques.

The terms “recombinant protein,” “heterologous protein” and “exogenousprotein” are used interchangeably to refer to a polypeptide which isproduced by recombinant DNA techniques, wherein generally, DNA encodingthe polypeptide is inserted into a suitable expression vector which isin turn used to transform a host cell to produce the heterologousprotein. That is, the polypeptide is expressed from a heterologousnucleic acid.

As used herein, the term “subject” can refer to any animal, including,but not limited to, humans and non-human animals (e.g., rodents,arthropods, insects, fish (e.g., zebrafish), non-human primates, ovines,bovines, ruminants, lagomorphs, porcines, caprines, equines, canines,felines, ayes, etc.), which is to be the recipient of a particulartreatment. Typically, the terms “patient” and “subject” are usedinterchangeably herein in reference to a human subject.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” mean theadministration of a subject supplement, composition, therapeutic orother material such that it enters the patient's system and, thus, issubject to metabolism and other like processes, for example,subcutaneous administration.

The phrase “therapeutically effective amount” means that amount of abioactive agent that, when present in a microsphere, produces somedesired effect at a reasonable benefit/risk ratio applicable to anymedical treatment. In certain embodiments, a therapeutically effectiveamount of a bioactive agent for in vivo use will likely depend on anumber of factors, including: the rate of release of the bioactive agentfrom the microsphere, which will depend in part on the chemical andphysical characteristics of the such microsphere, the identity of thebioactive agent, the mode and method of administration; any othermaterials incorporated in the microsphere in addition to the bioactiveagent.

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of any condition or disease.

Embodiments described herein relate to compositions that can be used forbioactive agent delivery and tissue engineering, and more particularlyto compositions that can provide delivery of bioactive agents in atemporally controlled or predetermined manner to cells or tissue in avariety of biomedical applications, including tissue engineering, drugdiscovery applications, and regenerative medicine.

In some embodiments, the composition can include a plurality ofparticulate coated hydrogel microparticles. Each of the microparticlescan include a hydrogel inner core and a porous particulate shell definedby a plurality of solid nanoparticles. The particulate shell can inhibitaggregation of the microparticles in an aqueous medium. Optionally, thehydrogel inner core can include one or more bioactive agents, and theparticulate shell can allow release or diffusion of the bioactive agentsfrom the hydrogel inner core in a sustained, controlled, and/orpredetermined manner.

The particulate coated hydrogel microparticles can be formed using areverse Pickering emulsion process that assembles the solid particlessuch that they are adsorbed onto outer surfaces of hydrogelmicroparticles of an emulsion. A “reverse Pickering emulsion” or a“water in oil Pickering emulsion” refers to an emulsion that utilizessolid particles as a stabilizer to stabilize droplets of a water solubleorganic substance, such as the gel forming natural polymer macromersdescribed below, in a dispersed phase in the form of microdroplets ormicroparticles dispersed throughout a continuous phase, which comprisesan organic solvent or oil medium. As used herein, the term “adsorbed”refers to the adherence of atoms, ions, or molecules of a gas or liquidto the surface of another substance by a relatively small force, such asa force on the order of van der Waals forces, as opposed to a chemicalreaction or covalent bond.

Advantageously, it was found that particulate coated hydrogelmicroparticles, which include a gelatin core and nanoparticle shell canoutperform traditional gelatin microparticles both in supporting cellfunctions and releasing drugs. In addition, due to its ability of easilysuspending in aqueous solution, the particulate coated hydrogelmicroparticles can be readily adopted for large scale production oftissue constructs for tissue engineering applications and for generatingcell aggregates for in vitro testing of pharmaceutical agents.

In some embodiments, the hydrogel inner core can include a naturalpolymer macromer that is soluble in an aqueous solution and can form ahydrogel upon gelation. The natural polymer macromer can besubstantially cytocompatible (i.e., substantially non-cytotoxic) andhave controllable physical properties, such as degradation rate,swelling behavior, and mechanical properties. Examples of naturalpolymer macromers include collagen, gelatin, glycosaminoglycans (GAG),poly (hyaluronic acid), alginate, hyaluronan, agarose,polyhydroxybutyrate (PHB), and combinations thereof.

The natural polymer macromers used to form the hydrogel microparticlesmay be cross-linked with a cross-linking agent in order to enhance themechanical strength of the microparticles and/or control physicalproperties of the microparticles, such as degradation rate, swellingbehavior. Examples of cross-linking agents may include divalent cations,genipin, glutaraldehyde, tri-polyphosphate (TPP), hydroxyapitite (HA),and any other cross-linking agent known to those skilled in the art. Byway of example, the natural polymer macromer can include gelatin

The natural polymer macromer (e.g., gelatin) can also be acrylatedand/or methacrylated by reacting an acryl group and/or methacryl anatural polymer macromer (e.g., gelatin). For example, bovine type-Bgelatin can be dissolved in a phosphate buffered solution and thenreacted with methacrylic anhydride to provide a plurality ofmethacrylate groups on the gelatin.

The degree of acrylation and/or methacrylation can be controlled tocontrol the degree of subsequent crosslinking of the acrylate andmethacrylates as well as the mechanical properties, and biodegradationrate of the gelatin. The degree of acrylation or methacrylation can beabout 1% to about 99%, although this ratio can vary more or lessdepending on the end use of the composition.

In some embodiments, the acrylate or methacrylate groups of theacrylated and/or methacrylated natural polymer macromer can becrosslinked by photocrosslinking using UV light in the presence ofphotoinitiators. For example, acrylated and/or methacrylated naturalpolymer macromers can be photocrosslinked in an appropriate amount ofdiH₂O or aqueous media (e.g., PBS) containing a desired amount of aphotoinitiator.

The photoinitiator can include any photo-initiator that can initiate orinduce polymerization of the acrylate or methacrylate macromer. Examplesof the photoinitiator can include camphorquinone, benzoin methyl ether,2-hydroxy-2-methyl-1-phenyl-1-propanone,diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, benzoin ethyl ether,benzophenone, 9,10-anthraquinone, ethyl-4-N,N-dimethylaminobenzoate,diphenyliodonium chloride and derivatives thereof.

The acrylated and/or methacrylated natural polymer macromers can beexposed to a light source at a wavelength and for a time to promotecrosslinking of the acrylate and/or methacrylate groups of themacromers.

In some embodiments, microspheres generally have one or more dimensionsof less than about 1000 μm, for example less than about 500 μm, forexample, less than about 250 μm. In other embodiments, the hydrogelmicroparticles can have an average diameter of about 1 μm to about 500μm, about 25 μm to about 400 μm, or about 50 μm to 200 μm.

The solid nanoparticles used to define the porous particulate shellsurrounding the hydrogen inner core can include any solid nanoparticlethat can be readily adsorbed onto the interphase between two phases ofan emulsion, such an oil/water interface, to reduce overall interfacialenergy of the emulsion system and stabilize the emulsion. In someembodiments, the porous particulate shell comprises a monolayer ofinorganic nanoparticles. The inorganic nanoparticles can be metalnanoparticles, metal alloy nanoparticles, metal oxide nanoparticles, ora combination thereof.

The nanoparticles generally have one or more dimensions of less thanabout 1000 nanometers, for example less than about 500 nanometers, forexample, less than about 250 nanometers. For example, the nanoparticlescan have an average diameter of about 1 nm to about 999 nm. In someembodiments, the nanoparticles are spherical nanoparticles. In someembodiments, the nanoparticles are silica nanoparticles, siliconnanoparticles, gold nanoparticles, titanium oxide nanoparticles, and thelike, or a combination thereof. In other embodiments, the nanoparticlescomprise silica, titanium dioxide, or a combination thereof. In someembodiments, the inorganic nanoparticles comprise silica, for example,the inorganic nanoparticles can be silica nanoparticles. In someembodiments, the inorganic nanoparticles have an average diameter ofabout 1 nm to about 999 nm, about 50 nm to about 900 nm, about 75 nm to800 nm, or about 100 nm to 750 nm, or about 200 nm to about 700 nm.

The Pickering emulsion used to form the particulate coated hydrogelmicroparticles can be prepared by suspending a plurality of solidnanoparticles in an organic solvent, such as a plant derived oil, suchas vegetable oil or olive oil, that does not dissolve the hydrogenforming natural polymer macromers. The hydrogel forming natural polymermacromers are dissolved in an aqueous solution.

The aqueous solution of the hydrogel forming natural polymer macromersis then gently added to the organic solvent containing the solidnanoparticles. The added solution of natural polymer macromers formsdroplet in the organic solvent. The solid nanoparticles in the organicsolvent (e.g., oil) move to the organic solvent/water interface to forma reverse Pickering emulsion.

The mixture is agitated under shearing forces to reduce the size ofdroplets. During this time an equilibrium of the Pickering emulsion isreached and the size of the droplets is stabilized by the action of thesolid nanoparticles in coating the surface of the droplets of thenatural polymer macromer solution. The Pickering emulsion includes aplurality of uniformly dispersed microparticles of the hydrogel formingpolymer macromers and the solid nanoparticles coating outer surfaces ofthe hydrogel forming polymer macromers in a substantially continuouslayer. The substantially continuous layer means that the solidnanoparticles form a porous coating on the surface of the microparticlesor microdroplets of natural polymer macromer solution that issufficiently continuous enough to prevent coalescence of themicrospherers or microdroplets within the organic solvent.

The emulsion can then be gelated by, for example, reducing thetemperature of the system to the gelation temperature of the hydrogelforming polymer macromers. The hydrogel forming polymer macromers arethen solidified to form particulate coated hydrogel microparticles byextracting water from the emulsion. The water can be extracted from theemulsion by adding a third solvent that is miscible or substantiallymiscible with both the organic solvent and water and which does notdissolve the hydrogel forming polymer macromers. The particulate coatedhydrogel microparticles are then isolated from organic solvent byfiltration and washing with the filtered particulate coatedmicroparticles with the third solvent to remove the organic solvent andunbound solid particles.

In certain embodiments, the Pickering emulsions described herein aresubstantially free or, in some cases, completely free of any surfactant.As used herein, the term “surfactant” refers to materials that have anamphiphilic molecular structure, which includes a polar hydrophilicmolecular moiety and a nonpolar lipophilic molecular moiety, and whichacts to lower the interfacial tension between the dispersed phase andthe continuous phase in an emulsion. As will be appreciated, surfactantscan be classified as ionic (anionic, cationic, and amphoteric) ornonionic. As used herein, the term “substantially free” when used withreference to the absence of surfactant in the Pickering emulsionsdescribed herein, means that the emulsion comprises less than 0.05percent by weight of surfactant, based on the total weight of the solidparticle stabilizer and natural polymer macromers. As used herein, theterm “completely free” when used with reference to the absence ofsurfactant in the Pickering emulsions described herein, means that theemulsion comprises no surfactant at all.

In some embodiments, the particulate coated hydrogel microparticles caninclude one more bioactive agents that can be incorporated and/orencapsulated in the hydrogel inner core of the microparticles to providelocalized, sustained, and/or controlled release of the at least onebioactive agents to cells in or about the microparticles underphysiological conditions in a controlled or predetermined manner. Byincorporating and/or encapsulating bioactive agent in a microspheremicrodroplet, it is possible, in certain embodiments, to provide asteady dosage of such bioactive agent through a sustained or controlledrelease process. In addition, such encapsulation may protect thebioactive agent, or other materials from undesirable immunogenic,proteolytic or other events that would reduce the efficacy of thebioactive agent and may regulate immunogenic, proteolytic, or otherevents.

The at least one bioactive agent can include polynucleotides and/orpolypeptides encoding or comprising, for example, transcription factors,differentiation factors, growth factors, and combinations thereof. Theat least one bioactive agent can also include any agent capable ofpromoting tissue formation (e.g., bone and/or cartilage), destruction,and/or targeting a specific disease state (e.g., cancer). Examples ofbioactive agents include chemotactic agents, various proteins (e.g.,short term peptides, bone morphogenic proteins, collagen, glycoproteins,and lipoprotein), cell attachment mediators, biologically activeligands, integrin binding sequence, various growth and/ordifferentiation agents and fragments thereof (e.g., EGF), HGF, VEGF,fibroblast growth factors (e.g., bFGF), PDGF, insulin-like growth factor(e.g., IGF-I, IGF-II) and transforming growth factors (e.g., TGF-βI-III), parathyroid hormone, parathyroid hormone related peptide, bonemorphogenic proteins (e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13,BMP-14), sonic hedgehog, growth differentiation factors (e.g., GDF5,GDF6, GDF8), recombinant human growth factors (e.g., MP-52 and the MP-52variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-1,CDMP-2, CDMP-3), small molecules that affect the upregulation ofspecific growth factors, tenascin-C, hyaluronic acid, chondroitinsulfate, fibronectin, decorin, thromboelastin, thrombin-derivedpeptides, heparin-binding domains, heparin, heparan sulfate,polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interferingRNA molecules, such as siRNAs, DNA encoding for an shRNA of interest,oligonucleotides, proteoglycans, glycoproteins, and glycosaminoglycans.

The bioactive agents can be loaded, incorporated, and/or encapsulatedinto hydrogel microparticles during their preparation. For example, thebioactive agent can be initially combined with the hydrogel formingnatural polymer macromer prior to mixing with the organic solvent sothat the bioactive agent is provided in the hydrogel microspheres.Alternatively or additionally, the at least on bioactive agent can becombined with the particulate coated hydrogel microparticles afterformation and/or isolation by mixing the particulate coated hydrogelmicroparticles in a solution containing bioactive agent.

The amount of bioactive agent provided in the microparticles will dependon a number of factors, including: (i) the identity of the bioactiveagent; (ii) the microsphere's intended use, including any desiredtherapeutic effect for in vivo use; (iii) the chemical and physicalproperties of the microsphere, including the release rate ofencapsulated bioactive agent or other material under differentconditions.

In certain embodiments, a sufficient amount of the bioactive agent canbe incorporated into the microparticles to produce a therapeuticallybeneficial result. In those embodiments in which the bioactive agent isa polypeptide, such as BMP-2 or TGF-β, the polypeptide loaded in anymicrosphere may range from less than about 0.05 to more than about 50weight percent, or about 0.1, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0,5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, 40, or 45 weightpercent.

In addition to bioactive agents, other materials may be incorporatedinto the microparticles. Such additional materials may affect thetherapeutic and other characteristics of the microsphere that results.One example of such another material is an adjuvant. (Such materials mayalso be termed bioactive agents if appropriate.)

Alternatively, materials that augment the therapeutic effect of thebioactive agent may be incorporated into the microparticles. Forexample, natural polymers, such as heparin, that control and/or delaythe release of the bioactive agent can be provided in the microsphere.(Such materials may also be referred to as bioactive agents asappropriate). The amount of any such augmenting agent to be loaded intoany microsphere will depend on a variety of factors, including thenature of the such agent, the microsphere, whether there are any othermaterials incorporated in addition to the bioactive agent, and the like.For any such agent, the present invention contemplates incorporating asufficient amount to augment the therapeutic effect of the bioactiveagent. In other embodiments, the amount of such augmenting agent mayrange from about 0.005% up to about 25%, or alternatively 0.01, 0.05,0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 15 or 20%.

In some embodiments, more than two different bioactive agents can beloaded into the microparticles. In certain embodiments, three, four,five or more bioactive agents augmenting agents, fillers or othermaterials may be incorporated in any microsphere microdroplet and/orhydrogel.

The release rate of the bioactive agent from the microparticles microwill vary with different embodiments. For example, one subjectformulation may require at least an hour to release a major portion ofthe bioactive agent into the surrounding medium, whereas anotherformulation may require about 1-24 hours, or even much longer. Incertain embodiments, such release may result in release (over, say 1 toabout 2,000 hours, or alternatively about 2 to about 800 hours) of thebioactive agent or other material encapsulated in the microparticles. Incertain embodiments, such substance or other material may be released inan amount sufficient to produce a therapeutically beneficial response.

The release profile of any bioactive agent or other material from themicroparticles may vary in different embodiments. In one embodiment, thebioactive agent or other material is released from the microparticles ina pulsatile manner. For example, such a pulsatile manner may involverelease of the bioactive agent or other material in three phases: aninitial burst, a slow release, and a second burst. In anotherembodiment, the bioactive agent or other material is released in asustained manner. In still other embodiments, a significant portion ofthe bioactive agent or other material is released in an initial phase.In still other embodiments, the release profile is bi-phasic ormulti-phasic.

In some embodiments, solid particles of particulate shell cansubstantially prevent release of the bioactive agent from themicroparticles for a certain time period and then allow release of thebioactive agent thereafter, and the delayed release can be adjusted tothe time necessary for the compositions to reach the desired location.The solid particles that form the porous particulate shell can beconfigured or selected so that diffusion or release of the bioactiveagent from the inner core may be delayed or sustained.

Delayed release can be for a time of about 1 hr or greater, about 2 hrsor greater, about 4 hrs or greater, about 8 hrs or greater, about 12 hrsor greater, about 24 hrs or greater, about 2 days or greater, about 3days or greater, about 4 days or greater, about 5 days or greater, about1 week or greater, or about 2 weeks or greater. In each instance, themaximum time of delayed release can be about 3 weeks, about 4 weeks, orabout 6 weeks. In particular embodiments, delayed release can be a timeof about 1 hr to about 1 week, about 2 hrs to about 5 days, about 4 hrsto about 2 days, or about 8 hrs to about 24 hrs. Sustained release canbe calculated from the time bioactive agent release begins, from thetime of first delivery of the microparticles, or from the time that themicroparticles first encounter the conditions of the desired deliverylocation.

In some embodiments, release can be delayed as noted above and also besustained once release begins. Sustained release can proceed for a timeof about 12 hrs or greater, about 24 hrs or greater, about 2 days orgreater, about 3 days or greater, about 4 days or greater, about 5 daysor greater, about 1 week or greater, or about 2 weeks or greater. Ineach instance, the maximum duration of sustained release can be about 3weeks, about 4 weeks, about 6 weeks, or about 12 weeks. In particularembodiments, sustained release can be a time of about 12 hrs to about 6weeks, about 24 hrs to about 4 weeks, or about 2 days to about 2 weeks.

In other embodiments, the controlled release of the bioactive agent canbe controlled by controlling the degree or amount of cross-linking ofhydrogel inner core. For example, the hydrogel inner core of themicroparticles can be configured to one or more labile crosslinkinggroups. The crosslinking groups can maintain the hydrogel microsphereconfiguration until contact with a material and/or condition adapted tobreak the crosslinks.

In some embodiments, the particulate coated hydrogel microparticles canprovide localized, sustained, and/or controlled release of the at leastone bioactive agents to cells in or about the microparticles in acontrolled or predetermined manner. For example, a plurality of cellscan be mixed with hydrogel microparticles that include a bioactiveagent, and the bioactive agent can be controllably release in a spatialor temporal manner to facilitate proliferation, growth, and/ordifferentiation of the cells. The cells can include any cell, such asprogenitor cells, totipotent stem cells, a pluripotent stem cells, or amultipotent stem cells, differentiated cells, cancer cells as well asany of their lineage descendant cells, including more differentiatedcells (described above), such as MSCs and cancer stem cells.

The cells can be autologous, xenogeneic, allogeneic, and/or syngeneic.Where the cells are not autologous and are potentially administered to asubject for therapeutic applications, it may be desirable to administerimmunosuppressive agents in order to minimize immunorejection. The cellsemployed may be primary cells, expanded cells, or cell lines, and may bedividing or non-dividing cells. Cells may be expanded ex vivo prior tocombination or mixing with the hydrogel. For example, autologous cellscan be expanded in this manner if a sufficient number of viable cellscannot be harvested from the host subject. Alternatively oradditionally, the cells may be pieces of tissue, including tissue thathas some internal structure. The cells may be primary tissue explantsand preparations thereof, cell lines (including transformed cells), orhost cells.

Generally, cells can be combined with the particulate coated hydrogelmicroparticles in vitro, although in vivo seeding approaches canoptionally or additionally be employed. If the mixture of particulatecoated hydrogel microparticles and cells is to be implanted for use invivo after in vitro seeding, for example, sufficient growth medium maybe supplied to ensure cell viability during in vitro culture prior to invivo application. Once the mixture has been implanted, the nutritionalrequirements of the cells can be met by the circulating fluids of thehost subject.

Alternatively or additionally, cells may be layered on the particulatecoated hydrogel microparticles, or the particulate coated hydrogelmicroparticles may be added to a cell suspension and allowed to remainthere under conditions and for a time sufficient for the cells toincorporate within or attach to the microsphere hydrogel. Generally, itis desirable to avoid excessive manual manipulation of the cells inorder to minimize cell death. For example, in some situations it may notbe desirable to manually mix or knead the cells with the hydrogelmicroparticles; however, such an approach may be useful in those casesin which a sufficient number of cells will survive the procedure.

As those of ordinary skill in the art will appreciate, the number ofcells to be mixed with the particulate coated hydrogel microparticleswill vary based on the intended application of the hydrogel and on thetype of cell used. Where dividing cells are being introduced by mixingwith the particulate coated hydrogel microparticles, for example, alower number of cells can be used. Alternatively, where non-dividingcells are mixed with the particulate coated hydrogel microparticles, alarger number of cells may be required.

In another embodiment, particulate coated hydrogel microparticles maycontain particles useful to locate the microsphere for diagnosticapplications and the like. In certain embodiments, particulate coatedhydrogel microparticles may contain paramagnetic, superparamagnetic orferromagnetic substances which are of use in magnetic resonance imaging(MRI) diagnostics. For example, submicron particles of iron or amagnetic iron oxide may be incorporated into particulate shell orhydrogel inner core to provide ferromagnetic or superparamagneticparticles. Paramagnetic MRI contrast agents principally compriseparamagnetic metal ions, such as gadolinium ions, held by a chelatingagent which prevents their release (and thus substantially reduces theirtoxicity). In another embodiment, microsphere micro and/or nanodropletsand/or hydrogels may contain submicron particles, such as magnetic ironoxide, which permit the magnetic separation of microparticles. Otherlabeled compounds, such as radionucleides, e.g., ³H, ¹⁴C, ¹⁸F, ³²P,^(99m)Tc, and ¹²⁵I, may also be utilized for visualizing cells andtissues, to which microsphere micro and/or nanodroplets and/or hydrogelsmay be bound, by means of X-rays or magnetic resonance imaging. Theparticulate coated hydrogel microparticles may also contain in certainembodiments, ultrasound contrast agents, such as heavy materials, e.g.,barium sulphate or iodinated compounds, to provide ultrasound contrastmedia.

In still other embodiments, the particulate coated hydrogelmicroparticles may be conjugated to targeting molecules attached to thesurface of the particulate shell or the inner hydrogel core, such asmonoclonal antibodies that preferentially bind to a receptor or othersite of interest. In certain embodiments, such targeting may achievetargeted delivery in vivo of the particulate coated hydrogelmicroparticles. To attach targeting molecules to the surface of anyparticulate coated hydrogel microsphere, it may be necessary to provides linker molecules. Such linker molecules may be used to attachtargeting molecules. Alternatively, the constituents that form theparticulate hydrogel microsphere, e.g., hydrogel, solid particles,and/or bioactive agent, may contain functional groups that allow forattachment of targeting molecules.

The particulate coated hydrogel microparticles can be injectable and/orimplantable, and can be provided in a solution or carrier vehicle. Theparticulate coated hydrogel microparticles can be used in a variety ofbiomedical applications, including tissue engineering, drug discoveryapplications, and regenerative medicine and cancer therapy.

In one example, a composition comprising suspended chondrogenic cells,such as MSCs, and particulate coated hydrogel microparticles, whichinclude a growth factor, such as BMP-2 and/or or TGF-β, can be used in amethod to promote tissue growth in a subject. One step of the method caninclude identifying a target site. The target site can comprise a tissuedefect (e.g., cartilage and/or bone defect) in which promotion of newtissue (e.g., cartilage and/or bone) is desired. The target site canalso comprise a diseased location (e.g., tumor). Methods for identifyingtissue defects and disease locations are known in the art and caninclude, for example, various imaging modalities, such as CT, MRI, andX-ray.

The tissue defect can include a defect caused by the destruction of boneor cartilage. For example, one type of cartilage defect can include ajoint surface defect. Joint surface defects can be the result of aphysical injury to one or more joints or, alternatively, a result ofgenetic or environmental factors. Most frequently, but not exclusively,such a defect will occur in the knee and will be caused by trauma,ligamentous instability, malalignment of the extremity, meniscectomy,failed ACI or mosaicplasty procedures, primary osteochondritisdessecans, osteoarthritis (early osteoarthritis or unicompartimentalosteochondral defects), or tissue removal (e.g., due to cancer).Examples of bone defects can include any structural and/or functionalskeletal abnormalities. Non-limiting examples of bone defects caninclude those associated with vertebral body or disc injury/destruction,spinal fusion, injured meniscus, avascular necrosis, cranio-facialrepair/reconstruction (including dental repair/reconstruction),osteoarthritis, osteoschlerosis, osteoporosis, implant fixation, trauma,and other inheritable or acquired bone disorders and diseases.

Tissue defects can also include cartilage defects. Where a tissue defectcomprises a cartilage defect, the cartilage defect may also be referredto as an osteochondral defect when there is damage to articularcartilage and underlying (subchondral) bone. Usually, osteochondraldefects appear on specific weight-bearing spots at the ends of thethighbone, shinbone, and the back of the kneecap. Cartilage defects inthe context of the present invention should also be understood tocomprise those conditions where surgical repair of cartilage isrequired, such as cosmetic surgery (e.g., nose, ear). Thus, cartilagedefects can occur anywhere in the body where cartilage formation isdisrupted, where cartilage is damaged or non-existent due to a geneticdefect, where cartilage is important for the structure or functioning ofan organ (e.g., structures such as menisci, the ear, the nose, thelarynx, the trachea, the bronchi, structures of the heart valves, partof the costae, synchondroses, enthuses, etc.), and/or where cartilage isremoved due to cancer, for example.

After identifying a target site, such as a cranio-facial cartilagedefect of the nose, the composition comprising suspended chondrogeniccells, such as MSCs, and particulate coated hydrogel microparticles canbe administered to the target site. The composition comprising suspendedchondrogenic cells, such as MSCs, and particulate coated hydrogelmicroparticles can be prepared according to the method described above.

Next, the composition comprising suspended chondrogenic cells, such asMSCs, and particulate coated hydrogel microparticles may be loaded intoa syringe or other similar device and injected or implanted into thetissue defect. Upon injection or implantation into the tissue defect,the composition can form into the shape of the tissue defect.

After implanting the composition comprising suspended chondrogeniccells, such as MSCs, and particulate coated hydrogel microparticles intothe subject, the progenitor cells microparticles can migrate into thetissue defect, express growth and/or differentiation factors, and/orpromote chondroprogenitor cell expansion and differentiation.Additionally, the presence of the hydrogel microparticles in the tissuedefect may promote migration of endogenous cells surrounding the tissuedefect.

In another example, the particulate coated hydrogel microparticles canbe suspended with a plurality of cancer cells, such as cancer stemcells, for in vitro testing of pharmaceutical agents. The pharmaceuticalagent can include an agent that modulates the cancer cell growth orproliferation. The pharmaceutical can be released from the hydrogelcoated microparticles or administered to the cells from an externalsource.

The following example is for the purpose of illustration only and is notintended to limit the scope of the claims, which are appended hereto.

Example

Pickering emulsion is a kind of emulsion that utilizes solid particlesas the stabilizer. Particles are adsorbed onto the interface between thetwo phases of the emulsion, such as oil/water interface, to reduce theoverall interfacial energy of the system and thus stabilize theemulsion. Taking advantage of Pickering emulsion mechanism, we developeda solid particle shell/gelatin core microsphere for drug delivery. Inthis structure, gelatin core serves as the drug carrier as traditionalgelatin microsphere carriers do. The particle shell has two functions.The first function is to prevent aggregation of gelatin due to reducedinteractions of gelatin molecules among different microspheres.Secondly, unlike solid surface shells, particle shell is permeable,allowing payloads to penetrate inward and outward microspheres. Themicrosphere can be further crosslinked by a variety of crosslinkers,such as genipin and aldehyde to increase the stability duringincubation.

Preparation steps of the core/shell microspheres are schematicallysummarized in FIG. 1. Briefly, solid particles are firstly suspended inwarm organic solvents, such as vegetable oil, and gelatin is dissolvedin warm aqueous solution. Then gelatin solution is gently added intoorganic phase under stirring and gentle warming. After that,water-in-oil emulsion will form and solid particles will move towater/oil interface to form reversed Pickering emulsion by reducing theinterfacial energy of the system. The emulsion is then gelated byreducing temperature of the system to less than 15° C. due to thegelation ability of gelatin. Water is then extracted from the microgelsby adding a third solvent that can be mixed with both phases, such asacetone. Solidified microspheres are obtained by filtration and washingwith the third solvent to remove organic solvent and unbound solidparticles. Traditional Pickering emulsion is oil-in-water style. Incurrent process, the phases in the system are reversed and theoil-in-water emulsion are prepared through the same mechanism.

As an example, 600 nm silica nanoparticles (FIG. 2A) are used as thecoating materials to make a core/shell microsphere following the aboveprocedure. Obtained core/shell microsphere has an average size of 63±22μm. These microspheres are easily resuspended in aqueous solution by 5 svortexing (FIG. 2B, C). In contrast, gelatin microspheres withoutparticle shells are quite clumpy and difficult to be resuspended underthe same condition (FIG. 2B, D). It is noteworthy that silicananoparticles can be replaced by other types of solid particles at nanoor micron sizes. Properties of obtained core/shell microspheres can alsobe tailored by switching particle types or tuning their surfacefunctions.

To confirm the core/shell structure, silica nanoparticles arefluorescently labeled by fluorescein isothiocyanate (FITC) and gelatinis fluorescently labeled by tetramethylrhodamine isothiocyanate (TRITC).The preparation procedure of the microsphere is the same as the onewithout fluorescence labels. Obtained microspheres are observed by laserscanning confocal microscopy (LSCM). To better display the shellstructure, microspheres are loaded within two pieces of thin glass coverslips without spacers, so that microspheres are squeezed by the gravityof top cover slip. LSCM images (FIG. 3) clearly demonstrate that silicaparticles form a thin shell on the surface (green channel) and gelatincompose the core layer (red channel). The shell layer appears porous,i.e., particles are not tightly packed on the surface and gaps betweensilica clusters can be seen (FIG. 3B). This morphology indicates asufficient coating with good permeability so that microspheres arereadily separated in aqueous and drugs can be easily loaded or released.Microsphere cores are almost fully composed of gelatin and nearly noparticles reside at the center of microspheres (FIG. 3c ), indicatingsimilar loading capability of core/shell microspheres as traditionalgelatin microspheres.

Gelatin core of uncrosslinked microsphere dissolves at 37° C., making itideal for short term drug delivery purposes. For sustained delivery orlong term biological support, microspheres can be crosslinked to enhancetheir thermostability. Crosslinkers include genipin, aldehydes, phenolsand other protein crosslinking reagents. After crosslinking,microspheres can be evenly mixed with cells to extracellularly supportcell survival/differentiation 12 and sustainably release drugs, or toinduce cell differentiation in engineered tissue constructs. Forexample, after genipin crosslinking, core/shell microspheres arehomogenously mixed with human mesenchymal stem cells (hMSCs) at 0.6 mgor 1.5 mg per million cells. The mixture is transferred to V-bottomnonadhesive 96 well plates at the density of 250,000 cells per well with200 μL Chondrogenic induction media. Transforming growth factor beta 1(TGF-β1) is exogenously supplied at 10 ng/mL. After the formation ofaggregates, cells are continuously cultured for 2 weeks with mediachanging every 2 days. Cells without microspheres or with similar sizedgelatin microspheres but without particle coating are cultured under thesame condition as controls. After 2 weeks, aggregates are collected forglycosaminoglycan (GAG) and DNA assays to determine chondrogenicdifferentiation extent. As shown in FIG. 4, hMSCs mixed with core/shellmicrospheres expressed significantly higher level of GAG. DNA assayresult indicates that cell numbers among different groups remainsimilar. After normalization of GAG level to DNA amount (GAG/DNA ratio),average GAG production level of cells mixed with core/shell microspheresis significantly higher than controls. Obviously, core/shell microspherebetter supports hMSC differentiation than gelatin microsphere withoutparticle shell or hMSC alone does. This improvement is presumably due tobetter cell-microsphere contact as a result of more homogenousintegration of core/shell microspheres to cell constructs.

After loading drugs, core/shell microspheres can serve as a long termdrug carrier. For example, after loading TGF-β1, core/shell microspheresare mixed with hMSCs as endogenous supplier of TGF-β1 for chondrogenicdifferentiation. Culture condition is similar to above but withoutexogenous support of TGF-β1. Cells without any microspheres or with samesized gelatin microspheres without particle shells were cultured underthe same condition as controls. After 2 weeks culture, hMSCs mixed withcore/shell microspheres expressed significantly higher level of GAG.After normalization with DNA amount, the GAG/DNA ratio is alsosignificantly higher than controls. This result indicates that particleshell significantly enhances the long term drug supply performance ofgelatin microspheres.

In summary, a novel gelatin core/solid particle shell microspherestructure is invented. This invention harnesses Pickering emulsionmechanism, assembles solid particles onto surfaces of gelatin emulsionthrough reverse Pickering emulsion formation process. Gelatincore/particle shell microspheres outperform traditional gelatinmicrospheres both in supporting cell functions and releasing drugs. Inaddition, due to its ability of easily suspending in aqueous solution,core/shell microspheres can be readily adopted for large scaleproduction of tissue constructs. This technique will be particularlyinteresting to tissue engineering companies to make in vitro engineeredtissues, and pharmaceutical companies to generate cell aggregates for invitro testing of drugs mimicking 3D cellular environment.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

Having described the invention, we claim:
 1. A composition comprising: aplurality of particulate coated hydrogel microparticles, each of themicroparticles including a hydrogel inner core and a particulate shelldefined by a plurality of solid nanoparticles, the particulate shellinhibiting aggregation of the microparticles in an aqueous medium andbeing permeable to allow release of bioactive agents provided in thehydrogel inner core from the hydrogel inner core.
 2. The composition ofclaim 1, wherein the microparticles have an average diameter of about 1μm to about 500 μm.
 3. The composition of claim 1, wherein the hydrogelinner core comprises gelatin.
 4. The composition of claim 3, wherein thegelatin is at least partially cross-linked.
 5. The composition of claim1, wherein the nanoparticles have an average diameter of about 1 nm toabout 999 nm.
 6. The composition of claim 1, wherein the plurality ofsolid nanoparticles include solid inorganic nanoparticles
 7. Thecomposition of claim 6, wherein the solid nanoparticles include silicananoparticles.
 8. The composition of claim 1, wherein the hydrogel innercore includes at least one bioactive agent.
 9. The composition of claim1, further comprising a plurality of cells, the cells being dispersedwith the microparticles in the composition.
 10. A method of a forming aplurality of particulate coated hydrogel microparticles; the methodcomprising: suspending a plurality of solid nanoparticles in an organicsolvent; dissolving hydrogel forming natural polymer macromers in anaqueous solution; adding the aqueous solution of the hydrogel formingnatural polymer macromers to the organic solvent containing the solidnanoparticles to form a Pickering emulsion, wherein the Pickeringemulsion includes a plurality of uniformly dispersed microparticles ofthe hydrogel forming polymer macromers and the solid nanoparticlescoating outer surfaces of the hydrogel forming polymer macromers;solidifying the hydrogel forming polymer macromers to form particulatecoated hydrogel microparticles; and isolating the particulate coatedhydrogel microparticles from the organic solvent.
 11. The method ofclaim 10, wherein each of the microparticles includes a hydrogel innercore and a particulate shell defined by a plurality of solidnanoparticles, the particulate shell inhibiting aggregation of themicroparticles in an aqueous medium and being permeable to allow releaseof agents from the hydrogel inner core
 12. The method of claim 10,wherein the organic solvent comprises a plant derived oil.
 13. Themethod of claim 10, further comprising extracting water from theparticulate coated hydrogel microparticles.
 14. The method of claim 10,wherein the microparticles have an average diameter of about 1 μm toabout 500 μm and the nanoparticles have an average diameter of about 1nm to about 999 nm.
 15. The method of claim 10, wherein particulatecoated hydrogel microparticles are isolated from the organic solvent byfiltration.
 16. The method of claim 10, wherein the hydrogel formingpolymer macromer comprises gelatin.
 17. The method of claim 16, whereinthe gelatin is at least partially crosslinked.
 18. The method of claim10, wherein the plurality of solid nanoparticles include solid inorganicnanoparticles
 19. The method of claim 18, wherein the solidnanoparticles include silica nanoparticles.
 20. The method of claim 11,wherein the hydrogel inner core includes at least one bioactive agent,the at least one bioactive agent being added to the hydrogel inner coreduring or after formation of the particulate coated hydrogelmicroparticles.